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Int J Clin Exp Med 2014;7(12):4877-4886 www.ijcem.com /ISSN:1940-5901/IJCEM0003574

Original Article Effect of adenoviral delivery of gene on experimental inflammatory pain induced by formalin in rats

Xionggang Chen1, Tingting Wang2, Caizhu Lin1, Baihong Chen1

1Department of Anesthesiology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, P. R. China; 2Department of Ophthalmology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, P. R. China Received November 3, 2014; Accepted November 13, 2014; Epub December 15, 2014; Published December 30, 2014

Abstract: Circumstantial evidences suggest that and their common precursor prodynorphin (PDYN) are involved in antinociception and neuroendocrine signaling. DREAM knockout mice had increased levels of PDYN and expression, and reduced sensitivity to painful stimuli. However, some data support the notion that the up-regulation of spinal dynorphin expression is a common critical feature in neuropathic pain. It is not clear whether the production of can be increased when more PDYN is present. In this study we investigated the changes in pain behaviors, spinal PDYN mRNA expression and dynorphin A production on formalin-induced pain in rats receiving the pretreatment of adenoviral delivery of PDYN. Our results showed that the adenoviral transfer of PDYN gene was sufficient to reduce pain behaviors resulting from formalin injection, and the antinociceptive effect after receiving the pretreatment of adenoviral delivery of PDYN was mediated at the level of the spinal cord via KOR.

Keywords: Adenoviral delivery, prodynorphin, dynorphin A, formalin, hyperalgesia, spinal dorsal horn

Introduction The endogenous kappa- system also participates in facilitation of spinal nociceptive It is currently accepted that the endogenous transmission [1, 13]. Several experimental kappa-opioidergic system plays an important models of chronic pain states show that a sig- role in the endogenous pain control system nificant upregulation of spinal dynorphin A and [1-4]. The endogenous ligands for kappa- its precursor , PDYN, is a common con- receptor (KOR) consist of prodynorphin (PDYN), sequence of nerve injury [14]. Previous obser- dynorphin A, , α-neo-endorphin and vations showed that the mechanism of signifi- [5], whereas PDYN is the precur- cant increase of PDYN expression in peripheral sor of dynorphins [6-8]. Actions of these pep- neuropathy was proposed to explain the para- tides are preferentially mediated through KOR dox of dynorphin actions [15]. Otherwise, a and are critical for regulation of nociceptive study with PDYN knock-out mice that up-regu- transmission [9, 10]. Otherwise, the endoge- lated spinal dynorphin was required for the nous kappa-opioidergic system also includes maintenance of persistent neuropathic pain downstream regulatory element antagonistic [16]. Recent findings implicate a direct excitato- modulator (DREAM) which inhibits transcription ry action of dynorphin A at receptors of the PDYN gene [11, 12]. In DREAM knockout and NMDA receptors to promote hyperalgesia mice, there was increased expression of PDYN in nerve injured rats, and its upregulation may mRNA, but endogenous dynorphin increases promote nociceptive input due to injury [17-22]. might be limited to within physiological ranges All these data support the notion that the up- [6]. Therefore, the expression of endogenous regulation of spinal dynorphin expression is a PDYN, dynorphins may be under the tight con- common critical feature of expression of neuro- trol of DREAM in central nerve system. pathic pain [23]. Prodynorphin and inflammatory pain

Previous study found that PDYN knockout mice LacZ-a (Microbix Biosystems, Inc.) were all almost didn’t produce dynorphin [24], indicat- digested with Hind III and EcoR I, and the ing that dynorphin cannot be produced without digested fragment s were re-collected from the PDYN. Dynorphin A is an endogenous opioid low melt agarose gel and fast ligated directly by derived from the PDYN gene [6, T4 DNA ligase. The reaction product was trans- 25]. However, we are not aware whether the formed into E. coli (DH5α), and the positive bac- production of dynorphin A is obviously increased terial colony was selected. The recombinant when more PDYN is present. To our knowledge, eukaryotic expression vector pDC316-PDYN the involvement of the pretreatment of PDYN in was identified by using restriction endonucle- inflammation-induced pain has not been previ- ases digestive reaction and DNA sequencing. ously investigated. Therefore in this study we The HEK 293 cells were cotransfected by the investigated the behavior change of rats receiv- shuttle plasmid of pDC316-PDYN and the skel- ing the pretreatment of adenoviral delivery of eton plasmid of pBHG which was adenoviruses PDYN, PDYN mRNA expression and dynorphin A type 5 with deletions in E1 and E3, and the production on Formalin-induced pain. We recombinant plasmid of Ad5-PDYN was ob- hypothesized that rats receiving the pretreat- tained. The expression of the transfected genes ment of adenoviral delivery of PDYN by the tail was evaluated by PCR and immunocytochemi- vein would show a modulatory antinociceptive cal staining. Virus was purified by double CsCl action and dynorphin A down-regulation in the centrifugation and subsequently dialyzed as spinal cord. described previously [29, 30]. Final yields as assessed by plaque assays on 293 cells were 12 Materials and methods approximately 1 × 10 plaque forming units (pfu)/ml [31].

Experimental animals Recombinant adenoviral injection

Adult male SPF grade Sprague-Dawley rats Group Ad5 received respectively Ad5, with 1 × weighing between 250 and 300 g were housed 1011 pfu in 100 µl of PBS by the tail vein in day at a constant ambient temperature of 24°C ± 1 and day 7; group Ad5-PDYN received respec- 1°C, humidity of 40%-70% under a 12 h light/ tively Ad5-PDYN, with 1 × 1011 pfu in 100 µl of dark cycle and given food and water ad libitum. PBS by the tail vein in day 1 and day 7, while The rats were individually housed in plastic group C were injected with normal saline as cages with wood-chip bedding for at least 1 day control. Infusions of the recombinant adenovi- before surgery. All experimental procedures ruses into the tail vein of rats anesthetized with were approved by the Institutional Animal Care pentobarbital sodium (50 mg/kg, i.p.) were per- and Use Committee of First Affiliated Hospital formed as reported previously [32]. of Fujian Medical University and were in accor- dance with the guidelines for the use of labora- Experiment 1: The SD rats were randomly divid- tory animals [26]. All efforts were made to mini- ed into three groups (n = 10): group C, group mize animal suffering and to reduce the num- Ad5 and group Ad5-PDYN to assess whether ber of animals used. adenoviral delivery of prodynorphin inhibits experimental inflammatory pain induced by for- Recombinant adenoviral vectors malin in rats. Experiment 2: The SD rats were randomly divid- Construction of the recombinant adenoviruses ed into group Ad5 (n = 5) and group Ad5-PDYN was determined as described previously [27]. (n = 10) to determine whether the possible role Briefly, PDYN gene (GenBank accession num- of nor- (nor-BNI) after receiving ber. NM 019374) primer sets (Sence: the pretreatment of adenoviral delivery of 5’-GACATGGCGTGGTCCAGGCTGATG-3’; Antisen- PDYN, and nor-BNI was dissolved in artificial ce: 5’-GTCTCAAACATCTAAATCT TCAGAATAGG- cerebrospinal fluid (aCSF) [33]. Rats in the con- TATTGGG-3’) were designed via PDYN cDNA trol group received aCSF infusions. We used was cloned by PCR technique, and then insert- the method of intrathecal injection to infuse ed into the cloning vector pUC57. The recon- Nor-BNI or aCSF [34]. Intrathecal injection (i.t.) structed plasmid with PDYN gene was detected was given in a volume of 5 µl through an inter- by the electrophoresis and the sequence analy- vertebral space at the level of the 5th or 6th sis [28]. Plasmid pUC57-PDYN and pDC316- lumbar vertebra using a microsyringe.

4878 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain

Formalin-induced nociceptive behaviors and se: 5’-CACCCGCGAGTACAACCTTC-3’; antisense: pain intensity scoring 5’-CCCATACCCACCATCACACC-3’) were designed and synthesized by Invitrogen. PCR was per- On day 6 after second virus injection, the rats formed after reverse transcription at 42°C for were placed in a transparent glass trough and 60 min and initial denaturation at 95°C for 10 were allowed to get habituated for about 30 min. The temperature cycles (Roche, Bran- min. Then formalin (50 µl, 5%) was injected sub- chburg, NJ) were 94°C/30 s (denaturing), cutaneously (s.c.) into the large lateral footpad 58°C/30 s (annealing), and 72°C/30 s (exten- on the plantar surface of the right hind paw by sion). A total of 32 cycles and a final 5-min 100 µl microsyringe. Each rat was measured extension at 72°C were conducted. The PCR 12 times and Formalin-induced nociceptive amplified fragments, 239 bp for PDYN and 207 behaviors of rats were observed by trained indi- bp for β-actin, were separated on 3% ethidium viduals who were blinded to experimental con- bromide stained agarose gel (Invitrogen, ditions, and continued for the next 60 min [35]. Carlsbad, CA). The PCR gel image was captured Pain intensity scoring (PIS) of nociceptive be- and analyzed by a gel documentation system havior was recorded per 5 min by assigning (BIO-RAD, USA). The positive PCR bands were weights to the following categories: 0 = the purified and sequenced, and the resulting injected paw is no abnormalities; 1 = no or little sequences were identical to the targeted cDNA weight is placed on the injected paw (weight = sequences. Each experiment was run three 1); 2 = the injected paw is elevated and not in times and each sample was run in triplicate. contact with any other surface (weight = 2); 3 = the injected paw is licked, bitten or shaken Radioimmunoassay of dynorphin A content (weight = 3) [36]. PIS for each animal was calcu- lated by the total test session period (300 s) The spinal tissue was collected in the same way using the following formula. PIS = (Tl + 2 × T2 + as the procedure of RT-PCR. Radioimmunoassay 3 × T3)/300 T1, T2, T3 represents the time an was performed for quantitative determination animal spent in each behavioral category [37]. of dynorphin A at the protein level in the spinal Reverse transcription-polymerase chain tissue [40]. The tissue homogenization was reaction prepared as described previously [41]. Briefly, tissues were homogenized, and samples were After PIS testing at day 13, animals were rapidly spun at 3000 × g for 30 min at 4°C. The aliquot sacrificed by decapitation. The L4-6 lumber spi- supernatant was stored at -80°C for the subse- nal cord (n = 6 per group) was rapidly removed, quent protein quantification. Dynorphin A was and immediately deep-frozen in liquid nitrogen eluted and analyzed by radioimmunoassay [42, and stored at -80°C for further analysis. RT-PCR 43]. Rabbit anti-dynorphin A antibodies were was used to determine effects of recombinant generated against peptide C-terminus and adenoviral vector on PDYN mRNA expression demonstrated 100% molar cross-reactivity [38]. The total RNA was extracted from L4-6 with dynorphin A. Results were expressed and lumber spinal cord using Trizol reagent (Life analyzed as fmol/mg wet tissue weight, and the technologies). Purified RNA was treated with protein concentrations were determined by the RQ1 RNase-free DNase (Promega, 1 units/2 µg Bradford assay [44]. of total RNA) and reverse transcribed with SuperScript II Reverse Transcriptase (Invitro- Immunohistochemistry gen) according to the manufacturer’s protocol. Both concentration and purity of the total RNA After PIS testing at day 13, the animals (n = 4) were determined through measuring the absor- while deeply anesthetized with pentobarbital bance at 260 and 280 nm by spectrophotome- (50 mg/kg, i.p.), were perfused transcardially try [39]. The cDNA sequences of rat PDYN (No. through the ascending aorta with normal saline, NM_019374) and β-actin (No. NM_031144) immediately followed by 4% paraformaldehyde were obtained from GenBank at the National (0.1 mol/L phosphate buffer, pH 7.4). For histo- Center for Biotechnology Information (NCBI). pathological analysis of the spina1 cords, the Specific primer sequences for PDYN (sense: L4-6 spinal cord segments were removed and 5’-GCTCACAGAACTGCCATAGG-3’; reverse: 5’-C- post-fixed in 4% paraformaldehyde at 4°C, to CCATACCCACCATCACACC-3’) and β-actin (sen- remove water in the tissue. Paraffined sections

4879 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain were selected for further immunohistochemical processing. A half of sections were tested by Hematoxylin and Eosin (H&E) histology [45]. The sections were examined using a light micro- scope and were photographed.

Nor-BNI challenge

To determine whether the analgesic mecha- nism after receiving the pretreatment of adeno- viral delivery of PDYN in experiment 2, we injected nor-BNI (3 mg/kg) [33, 46], a specific kappa- antagonist (Tocris Bio- science, Ellisville, MO, USA), to five rats in group Ad5-PDYN before 2 h of PIS testing, while Figure 1. Pain intensity scoring (PIS) of three groups (n = 10 per group) in the formalin test. The formalin another five rats in group Ad5-PDYN were tests were performed after the injection of Ad5-PDYN injected with aCSF as control. Pain behavior in or the control viruses (Ad) twice by tail vein. After in- formalin test was measured. In order to rule out jection of Ad5-PDYN, there was a significant reduc- non-specific effects on pain processing of the tion of the nociceptive response in the second phase antagonist, rats in group Ad5 were also injected of the formalin test. *P < 0.05, **P < 0.01 versus group Ad5. with the same dose of nor-BNI.

Data analysis

Investigators were blinded to all treatments in all tests. Animals were randomly assigned to each group. SPSS 11.5 for windows (Chicago, IL, USA) was used for all data analyses. All data were presented as mean ± standard error of mean (SEM). Changes within each group over time were analyzed by using one-way ANOVA, and followed by post hoc comparison (Student- New-man–Keuls test). Significant differences between treatment groups were detected by two-way ANOVA. P < 0.05 is set as the level of statistical significance.

Results

Physiological functions

In the entire observation period, group Ad5- Figure 2. Effect of the pretreatment of adenoviral PDYN consumed similar amount of food and delivery of PDYN on Formalin test (n = 6 per group). fluid compared with group C, group Ad5, respec- A. An example of agarose gel electrophoresis of PCR tively (P > 0.05). No significant difference was products. β-actin PCR (a specific 207-bp segment of cDNA) was used as an internal control. B. Quantifica- observed in terms of body weight, rectal tem- tion of relative levels of spinal PDYN mRNA expres- perature, and respiratory rate among group C, sion. The PDYN mRNA levels of spinal cord in group group Ad5 and group Ad5-prodynorphin (P > Ad5-PDYN were markedly higher than those of spinal 0.05). cord in group Ad5. *P < 0.01 versus group Ad5.

Ad5-PDYN reduced pain behavior in the late phase of formalin test tored. Phase 1 of the agitation response was transient and lasted for approximately 10 min, After the right intraplantar injection of formalin, and no significant difference in pain intensity rats of three groups all evoked a biphasic agita- scoring (PIS) was found among group C, group tion response when the response was moni- Ad5 and group Ad5-prodynorphin (P > 0.05). As

4880 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain

and hyperemia were observed in spinal grey matter of Ad5-treated rats, but was not detect- ed in that of Ad5-PDYN-treated rats (Figure 4).

Nor-BNI challenge

In experiment 2, the anti-nociceptive effects of group Ad5-PDYN were antagonized by injection of Nor-BNI before 2 h of PIS testing, while no effect occurred with aCSF (Figure 5). No animal showed abnormal sensory or motor function after administration of Nor-BNI in group Ad5. Figure 3. Decreased dynorphin A protein in the spi- nal cord of rats treated with Ad5-PDYN. Dynorphin A Discussion protein of spinal cord in group Ad5-PDYN was signifi- cantly lower than those of spinal cord in group Ad5. In our behavioral studies we found that the ade- *P < 0.05 versus group Ad5. noviral transfer of the PDYN gene into tail vein was sufficient to reduce pain behavior resulting shown in Figure 1, phase 2 was sustained and from formalin test. The application of adenovi- lasted 50 min, PIS of group Ad5-prodynorphin ral vectors had previously been used to pro- was significantly lower than that of group Ad5 duce analgesia [47, 48] but these studies dif- (*P < 0.05, **P < 0.01), while no significant dif- fered from the present study in two key aspects. ference in PIS was found between group C and Firstly, in the current study gene transfer of group Ad5 (P > 0.05). PDYN by recombinant adenovirus attenuated inflammatory pain in rats; and secondly, the Increased PDYN mRNA in the spinal cord of antinociceptive effect after receiving the pre- rats treated with Ad5-PDYN treatment of adenoviral delivery of PDYN was mediated at the level of the spinal cord via kap- Rats treated with Ad5-PDYN displayed signifi- pa-opioid receptor. cant up-regulation in the expression of PDYN mRNA (9.47 ± 1.21) compared with group Ad5 We observed a significant antinociceptive (5.46 ± 0.29; *P < 0.01) (Figure 2), while no effect of Ad5-PDYN treatment on pain in the significant difference in the expression of PDYN late phase, but not the early phase, of the rat mRNA was found between group C and group formalin test, suggesting that Ad5-PDYN treat- Ad5 (P > 0.05). ment might more predominately reduce pain caused by inflammatory response than acute Ad5-PDYN reduced the production of pain in the early phase. As the late phase of dynorphin A formalin test was related to primary afferent Rats receiving Ad5 injection were served as the fiber-mediated central sensitization [35, 49], it control. A group of three rats were sacrificed in seemed that Ad5-PDYN treatment might effec- the same time period and the spinal cord was tively inhibit inflammatory-induced hyperactivi- obtained for dynorphin A radioimmunoassay. ty or central sensitization. As shown in Figure 3, dynorphin A protein of Gene transfer to nociceptive neurons of the spi- spinal cord in group Ad5-PDYN (1.53 ± 0.17) was significantly lower than those of spinal cord nal cord is a promising approach to dissect in group Ad5 (2.15 ± 0.28) (*P < 0.05), while no mechanisms of pain in rodents, and is a poten- significant difference in dynorphin A was found tial therapeutic strategy for the treatment of between group C and group Ad5 (P > 0.05). persistent pain disorders such as neuropathic pain. A number of studies have demonstrated Ad5-PDYN reduced inflammation in the spinal transduction of the central nervous system cord using adenovirus by tail vein [32, 50], oral administration [51] and injection directly [48]. H-E staining was performed in spinal cord tis- Recombinant adenovirus serotype 5 in the ner- sues of Ad5-treated and Ad5-PDYN-treated vous system currently have several advantages mice. Formalin-induced evident vasodilatation over other vectors, including stable and safe

4881 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain

Figure 4. Histology in spinal cord tissues of Ad5-treated and Ad5-PDYN-treated mice. Formalin-induced evident va- sodilatation and hyperemia were seen in spinal grey matter of Ad5-treated mice (A), but wasn’t detected in that of Ad5-PDYN-treated mice (B). H-E staining (Scale bar: 100 µm).

lower than those of spinal cord in group Ad5, and the PDYN mRNA levels in group Ad5-PDYN were markedly higher than those in group Ad5, also suggesting dynorphin A down-regulation and PDYN up-regulation, consistent with previ- ous reports [58]. There are possible reasons for the observed low level of dynorphin A pro- tein when exogenous PDYN was applied. One is that the function of pro-protein convertases [10] from PDYN to dynorphin A may be down- regulated via both lysosome and proteasome systems [59] after the pretreatment of adenovi- ral delivery of PDYN; the other is that the pro- Figure 5. Effects of Nor-BNI on group Ad5-PDYN. Val- duction of dynorphin A may be decreased dur- ues are expressed as mean ± SEM (five rats in each ing the observations when Ad5-PDYN treat- group). P < 0.05 as compared to the animals injected with aCSF. ment reduces the nociceptive effect induced by formalin, since spinal dynorphin A up-regulation is a consequence of nociceptive pain [14, 60, gene expression [52, 53]. In the pre-experi- 61]. Our data provided clear evidence that the ment, we found PDYN mRNA up-regulation in antinociceptive effect after receiving the pre- the spinal cord after Ad5-PDYN injection by tail treatment of adenoviral delivery of PDYN is vein twice in normal rats. Previous finding mediated at spinal cord via KOR, only suggest- showed that gene expression remained stable ing that PDYN-derived acting on KOR up to 5~8 days after infusion of the recombi- are important in the control of inflammatory nant adenovirus serotype 5 [54, 55]. The pres- pain, because PDYN obviously may be pro- ent study confirmed that the PDYN mRNA levels cessed to multiple peptides that could elicit a of group Ad5-PDYN were markedly higher than those of group Ad5 in spinal cord on day 6 after behavioral effect. Consistent with this, a pro- second virus injection, suggesting the recombi- nounced impact of endogenous PDYN-derived nant adenovirus successfully transferred the peptides on nociception was mediated via KOR PDYN gene into the spinal cord, consistent with [5, 9], showed the same effects. previous reports [56, 57]. Dynorphin A, an endogenous opioid neuropep- Here we showed that dynorphin A protein of spi- tides derived from the PDYN gene [6], is thought nal cord in group Ad5-PDYN was significantly to play a role in activity-dependent processes,

4882 Int J Clin Exp Med 2014;7(12):4877-4886 Prodynorphin and inflammatory pain including synaptic plasticity and learning and the endogenous opioid peptides dynorphins in memory [62, 63]. As the transcriptional repres- uninjured mice: evidence with intrathecal sor, DREAM regulates a number of genes N-ethylmaleimide inhibiting dynorphin degra- including PDYN [64, 65], and DREAM knockout dation. Int Rev Neurobiol 2009; 85: 191-205. (KO) mice showed significantly enhanced PDYN [2] Wang Y, Gehringer R, Mousa SA, Hackel D, mRNA expression and memory 24 hours after Brack A and Rittner HL. CXCL10 controls in- training as measured by percent freezing, sug- flammatory pain via -containing macrophages in electroacupuncture. PLoS gesting that dynorphin A didn’t obviously upreg- One 2014; 9: e94696. ulated [58]; while PDYN knockout mice demon- [3] Chen ZY, Wang H, Xu W, Xu H and Fu X. Effect strated diminished age-associated impairment of intravenous general anaesthesia with epi- in spatial water maze performance, suggesting dural block on the expression of pre-endoge- dynorphin A expression significantly downregu- nous opioid peptide genes. J Int Med Res lated [43]. These opposing effects of endoge- 2014; 42: 765-772. nous kappa-opioidergic system suggest that [4] Kardon AP, Polgar E, Hachisuka J, Snyder LM, control of DREAM-PDYN-dynorphin message Cameron D, Savage S, Cai X, Karnup S, Fan CR, may be very important between dynorphin A Hemenway GM, Bernard CS, Schwartz ES, expression and pathological processes. This Nagase H, Schwarzer C, Watanabe M, Furuta T, has led to the notion that PDYN and dynorphin Kaneko T, Koerber HR, Todd AJ and Ross SE. Dynorphin acts as a neuromodulator to inhibit A were involved in antinociception when dynor- itch in the dorsal horn of the spinal cord. phin A increases were be limited to within physi- Neuron 2014; 82: 573-586. ological ranges [6]; otherwise, endogenous [5] Merg F, Filliol D, Usynin I, Bazov I, Bark N, Hurd dynorphin A appeared to be involved in chronic YL, Yakovleva T, Kieffer BL and Bakalkin G. Big allodynia and hyperalgesia when dynorphin A dynorphin as a putative endogenous ligand for increases were in the supraphysiological rang- the kappa-opioid receptor. J Neurochem 2006; es [66]. We are not aware whether DREAM 97: 292-301. inhibits or promotes the production of dynor- [6] Hauser KF, Aldrich JV, Anderson KJ, Bakalkin phin A when more PDYN is present. Further G, Christie MJ, Hall ED, Knapp PE, Scheff SW, studies to delineate the interaction of spinal Singh IN, Vissel B, Woods AS, Yakovleva T and DREAM-PDYN-dynorphin message in pain Shippenberg TS. Pathobiology of dynorphins in trauma and disease. Front Biosci 2005; 10: states might lead to better strategies for the 216-235. prevention of inflammation-induced pain. [7] Zhang RX, Li A, Liu B, Wang L, Xin J, Ren K, Qiao JT, Berman BM and Lao L. Electroa- Acknowledgements cupuncture attenuates bone-cancer-induced hyperalgesia and inhibits spinal preprodynor- We would like to thank Zhiyong Zhong for his phin expression in a rat model. Eur J Pain technical assistance. This work was supported 2008; 12: 870-878. by a grant from the key teacher fund of middle- [8] Corchero J, Avila MA, Fuentes JA and age of Fujian Medical University (No. JGG- Manzanares J. delta-9- 201313). increases prodynorphin and gene expression in the spinal cord of the rat. Disclosure of conflict of interest Life Sci 1997; 61: PL 39-43. [9] Wittmann W, Schunk E, Rosskothen I, Gaburro None. S, Singewald N, Herzog H and Schwarzer C. Prodynorphin-derived peptides are critical Address correspondence to: Dr. Tingting Wang, modulators of anxiety and regulate neuro- Department of Ophthalmology, The First Affiliated chemistry and corticosterone. Neuropsy- Hospital of Fujian Medical University, Fuzhou chopharmacology 2009; 34: 775-785. 350005, P. R. China. Tel: +86 0591 87981990; [10] Chen Y, Chen C and Liu-Chen LY. Dynorphin E-mail: [email protected] peptides differentially regulate the human kappa opioid receptor. Life Sci 2007; 80: References 1439-1448. [11] Reisch N, Engler A, Aeschlimann A, Simmen [1] Tan-No K, Takahashi H, Nakagawasai O, BR, Michel BA, Gay RE, Gay S and Sprott H. Niijima F, Sakurada S, Bakalkin G, Terenius L DREAM is reduced in synovial fibroblasts of pa- and Tadano T. Nociceptive behavior induced by tients with chronic arthritic pain: is it a suitable

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